Tropical malaria, caused by the parasite Plasmodium falciparum, is responsible for up to three million deaths each year. Since the parasite develops resistance against most clinically available drugs, novel antimalarial drugs are urgently needed. Glucose-6-phosphate dehydrogenase (G6PD) is a novel target for antimalarial drug design based on observations that humans with a genetic deficiency in this enzyme are protected against malaria. G6PD catalyses the initial step of the pentose phosphate pathway, yielding NADPH, an essential reducing equivalent to detoxify oxidative stress in red blood cells (RBCs). The malaria parasite is susceptible to oxidative stress in the RBC stage. Naturally occurring G6PD deficiency leads to a lack of reducing equivalents, an increase in oxidative stress, enhanced phagocytosis of parasite-infected RBCs, and, as a consequence, to a protection against malaria. NADPH in parasite-infected RBCs is generated by human G6PD but also by a parasite enzyme with G6PD activity, called P. falciparum glucose-6-phosphate-dehydrogenase-6- phosphogluconolactonase (PfGluPho). We hypothesize that inhibiting PfGluPho and, to a certain extent, human G6PD reduces the risk of developing malaria. So far, exploring G6PD and PfGluPho as antimalarial drug targets was limited by a lack of recombinant PfGluPho. Very recently our German team produced the first complete and functional recombinant PfGluPho. We now aim at using this recombinant, pure protein to identify inhibitors which could be potential candidates for novel and innovative antimalarial drugs. In AIM 1 we will produce human G6PD and PfGluPho in mg quantities. AIM 2 then uses these proteins in already established high-throughput screening assays to identify enzyme inhibitors. Compounds active in the low micromolar to nanomolar concentration range will be subject to detailed kinetic analyses on isolated enzymes. AIM 3 follows up on the identified inhibitors and assesses whether they impact P. falciparum growth and parasitemia without being cytotoxic in mammalian cells. Since G6PD deficiency is a proven principle against malarial parasites and since the parasite enzyme differs structurally and mechanistically from the human host enzyme, PfGluPho is an excellent drug target. We aim at identifying 2-3 lead compounds which are active in the nanomolar range without significant cytotoxicity, which can be used for further drug development. This may be a high risk approach since it is not guaranteed that high-throughput screening and follow-up assays identify a hit. However, we have already identified 164 compounds that inhibit bacterial G6PD, and we anticipate a similar hit rate for PfGluPho and human G6PD. This approach has the potential to generate high impact results. Malaria is the most deadly disease worldwide. The malaria parasite develops resistance to most of the currently available drugs. Thus, novel and innovative antimalarial drugs are desperately needed. PUBLIC HEALTH RELEVANCE: Tropical malaria is responsible for up to three million deaths annually. The malaria parasite Plasmodium falciparum develops resistances against most clinically available drugs. Novel antimalarial drugs are urgently needed. Glucose-6-phosphate dehydrogenase is a novel target for antimalarial drug design based on observations that humans with a deficiency in this enzyme are protected from malaria. We aim at identifying compounds that inhibit this enzyme both in malaria parasites and, to a certain extent, in humans. Our results may pave the way for the development of novel antimalarial drugs.